The identification of effective chemotherapy and surgery for patients with osteosarcoma has led to significant improvements in outcome. The determination of prognosis in osteosarcoma continues to be based on clinical staging systems. The identification of additional prognostic factors may allow stratification of therapy. Investigation of biological features related to chemotherapy resistance or response may identify prognostic factors. Advances in the understanding of the molecular basis of resistance to methotrexate, an agent routinely used in high doses for osteosarcoma treatment, will allow the identification of in vitro assessments which may predict response to this drug. Recently identified mutations and polymorphisms in the reduced folate carrier which is involved in methotrexate uptake may also be important determinants of tumor chemotherapy response and patient treatment related toxicity. Studies of these biological factors may allow prioritization of therapeutic strategies as newer agents which avoid antifolate resistance have been developed. The aim of this study is to investigate biological factors related to methotrexate resistance as predictors of prognosis in patients with osteosarcoma. Osteosarcoma tumor samples obtained from patients treated as part of Children's Oncology Group therapeutic studies will be assayed for changes in reduced folate carrier, dihydrofolate reductase, folylpolyglutamate synthetase and gamma-glutamyl hydrolase expression as well as functional assays for methotrexate uptake and polyglutamylation. The results of these assays will be correlated with the histologic response of the tumors to preoperative chemotherapy and patient survival in a large uniformly treated cohort of patients with newly diagnosed localized osteosarcoma to potentially identify these assays as prognostic factors. Osteosarcoma tumor samples and patient's normal cells will be screened for alterations in reduced folate carrier sequence. The presence of the reduced folate carrier host polymorphisms will be related to the toxicity observed following high-dose methotrexate administration and the time required to clear the methotrexate to determine their utility at predicting patient toxicity. The tumor specific mutations will be evaluated as prognostic factors. It is hoped that the information obtained through this proposal will allow the development of risk stratified therapy for osteosarcoma that takes into account patients' likelihood of therapy related toxicity and probability of response to new agents.